Trypanosoma cruzi is the etiologic agent of Chagas disease in humans which afflicts over 20 million people in South and Central America. Although it has been well documented that different T. cruzi isolates vary dramatically is their ability to infect and cause disease little is known of the mechanisms responsible for this variation. So, the overall goal of this research plan is to understand the events which modulate infectivity and virulence on T. cruzi. To achieve this goal the objective of this work is to understand the basic mechanisms used to regulate gene expression during the developmental cycle of the parasite. The experimental plan is designed to characterize the expression of the CUB2.65 gene of T. cruzi. The experiments in the first section (I) are focused on two approaches to identify the minimal 5'-flanking sequence able to direct accurate trans-splicing of CUB2.65 and pre-mRNA. One approach uses in vivo expressed bacteriophage T7 RNA polymerase to drive transcription of the template RNA from the T7 promoter. This has the advantage of uncoupling trans-splicing from transcription, which is poorly understood in T. cruzi. The second approach uses artificial polycistronic transcription units to achieve the same goal. This section is followed by a description of experiments designed to study the CUB2.65 5'-flanking sequences with respect to their ability to drive gene expression in the transient and stable transformation systems (section II). These studies will also lead to the development of genetic tools which can be used to characterize transcription and trans-splicing. Lastly, if the genetic experiments outline in this proposal proceed more rapidly than currently anticipated, experiments will be undertaken to characterize protein/DNA interactions within the CUB2.65, 5'-flanking regions.